PARENT AWARD R35GM119627 SUMMARY The objective of my laboratory is to characterize how molecular communication between bacteria and their animal hosts leads to specific and reproducible colonization. To accomplish this goal, the laboratory studies the V. fischeri-squid system. This system is advantageous because bacteria colonize through the natural route of infection, all animals are colonized within three hours of inoculation into the seawater, the bacteria can be subject to detailed genetic manipulation, and the precise site of infection can be imaged directly in the live animal host, allowing for cutting edge genetic and genomic approaches. Focusing on how squid are reproducibly colonized by the specific symbiont, to the exclusion of the millions of competing bacteria in seawater, has revealed key roles for bacterial aggregation and biofilm formation in promoting specific host- microbe interactions. Recently our global genetic screen to identify colonization factors revealed multiple novel biofilm activators and identified an orphan histidine kinase, which acts as a negative regulator of Syp biofilm development in vivo. To elucidate the pathways governing colonization and biofilm development in the animal host we will (1) dissect the molecular details of the novel histidine kinase pathway, (2) define at high temporal and spatial resolution the steps necessary for the establishment of this mutualistic relationship, and (3) characterize the role of new colonization factors using genetic and metabolomic approaches. Since the strategies used by V. fischeri to interact with its host are conserved in other host<microbe systems, our findings will be applicable to understand other beneficial associations as well as interactions between pathogenic bacteria and their hosts.